When diagnosed early, most cancers are more apt to be prevented from metastasizing, which gives rise to a higher survival rate for patients. However, high diversity of cancer-related factors contributes to the difficulty of early diagnosis. Active studies have been directed towards biomarkers that are available for the early diagnosis of cancer. One of the most interesting biomarkers is cancer cell RNA. For use in diagnosis, the RNA has been obtained from cancer cells separated from excised cancer tissues or from CTC (circulating tumor cells) in the bloodstream. However, the excision is accompanied by an operation that is invasive to the subject, and the finding of CTC is poor in effectiveness because it is too rare, e.g., one cells per 109 blood cells, to detect.
Most cells shed extracellular vesicles (EV) into body fluid. EV serves as a transporter carrying various materials including proteins, lipids, amino acids, etc. These substances have recently been revealed to be important factors that are indicative of properties of the EV sources, that is, the cells shedding the EV. Thus, newly increasing recognition has been given to the importance of EV. Particularly, cancer cells shed a number of vesicles containing metastasis factors. Cancer cell-shed vesicles are present at a higher level in blood and thus are easier to apply to the diagnosis of cancer, compared to CTC. The vesicles shed from cancer cells can, therefore, be used for early diagnosis of cancer in a non-invasive manner.
In addition to the small size of vesicles, micron-sized substances and blood cells other than vesicles makes it difficult to obtain information on cancer cells from vesicles. For example, the extraction of information about vesicular nucleic acids of cancer cells by performing PCR (polymerase chain reaction), a powerful tool of analyzing nucleic acids, such as RNA, is obstructed by DNAs of blood cells such as leucocytes, and by pigments of hemoglobin. It is predicted that information derived from vesicles, if isolated with the exclusion of these obstacles, would be useful in the early diagnosis of cancer.
However, no effective and accurate methods of isolating vesicles from body fluid have been developed, thus far. Centrifugation is representative of the isolation methods of vesicles developed thus far. Primarily, cells are, for example, separated from body fluid on the basis of density difference using a centrifugal separator. Then, centrifugation at higher speeds removes other microparticles and aqueous solutions. However, the centrifugation method is operated in multiple steps, requiring much labor. In addition, the method needs huge facilities as well as a large quantity of body fluid, and thus it is impossible to apply to practical diagnosis in which only a small amount of samples can be utilized. In addition, it takes at least 4 hours to complete the centrifugation method, so that it is not suitable for clinical diagnosis requiring rapidity. A method for isolating microvesicles from body fluid using a microchip onto which an antibody to vesicular proteins is immobilized was developed (US 20120142001 of Skog et al., entitled “Method for isolation of nucleic acid containing particles and extraction of nucleic acids therefrom”), by which vesicles can be isolated within a short time. However, this method requires a centrifugation procedure for cell removal. A method that is capable of isolating vesicles from a small quantity of body fluid without using complex facilities such as centrifugation separators or without requiring the immobilization of antibodies to collection channels is advantageous in application to clinical diagnosis.
Leading to the present invention, intensive and thorough research into the isolation of vesicles has resulted in developing a microfilter system that is capable of effectively isolating microvesicles in a non-invasive manner and in finding that the microfilter system is applicable to the clinical diagnosis of cancer.